2.2 DNA extraction, RADseq library preparation and sequencing
Genomic DNA was extracted from each whole specimen using a DNeasy Blood & Tissue Spin Column Kit (Qiagen, Venlo, Netherlands), including the RNase treatment suggested by the manufacturer. The extraction was performed in whole specimens previously washed with sterilised water three times to remove any external contaminant in a room never exposed before to medflies. The DNA was quantified using Qubit 2.0 dsDNA HS Kit (Thermo Fisher Scientific, Waltham, US-MA) and sent to GenePool laboratories (Edinburgh, Scotland) for RAD-tag library preparation and sequencing. Briefly, DNA samples were digested using SbfI  high-fidelity restriction enzyme (NEB Inc., Ipswich, US-MA). P1 adapters, each with a unique eight bp molecular identifying sequence, were ligated using T4 DNA Ligase (NEB) to allow 16 individuals per lane multiplexing. Fragments were pooled and size selected (300-700 bp) prior to purification with Qiagen columns. Fragment ends were repaired using the Quick Blunting Kit (NEB), and P2 adapters were ligated using T4 DNA Ligase. Minelute columns (Qiagen) were used to purify DNA. PCR enrichment of the libraries was performed using Phusion Flash High-Fidelity PCR Master Mix (NEB) and size selected using gel electrophoresis. The sequencing of RAD tags was done on Illumina GAIIx (Illumina Inc., San Diego, US-CA) following standard protocols, and data is available via CSIRO Data Collection (Elfékih, Arias, & Vogler, 2020)